PROJECT SUMMARY/ABSTRACT The long-term objectives of this project are to increase our understanding of eukaryotic transcription elongation. The focus of the proposed experiments is the analysis of two essential transcription elongation factors named Spt5 and Spn1 (also known as Iws1). Both factors have been implicated in human health. Spt5 is required for HIV gene expression, expression of NF-?B-activated genes, and it is required for immunoglobulin class switching in B cells. Spn1 has been implicated in cancer. For both factors, there are several important areas where little is understood. Preliminary studies of Spt5 in the model system, S. pombe, have provided strong evidence that Spt5 is required for normal levels of transcription elongation genome-wide, with Spt5 required for RNA polymerase II to elongate past a barrier. These studies have also shown that Spt5 represses a novel class of antisense transcript that initiates at or near the barrier and that is synthesized across the 5' ends of the majority of genes. The proposed experiments in Specific Aim 1 address three related areas, continuing to use S. pombe as a model system. In Aim 1.1, two genome-wide approaches (MNase-seq and TSS-seq), will be employed to (1) test whether Spt5 controls chromatin structure and (2) to map the 5' ends of the antisense RNAs as a way to localize the antisense promoters and barriers with respect to nucleosome position. The results will provide new and comprehensive characterization of the role of Spt5 in transcription and chromatin structure. Aim 1.2 will focus on the sequences that have three possible functions: the barrier to elongation, the antisense promoter, and a possible site that stimulates elongation. Constructs will be made and tested to define the sequences required for these functions. The results will provide new insights into a previously unstudied aspect of eukaryotic transcription elongation. Aim 1.3 addresses the Spt5 protein itself, focusing on the isolation of new spt5 mutations that impair elongation. The positions and defects in the mutant proteins will reveal new understanding of Spt5 protein function. Specific Aim 2 proposes experiments in S. cerevisiae to understand the transcription factor Spn1. Aim 2.1 proposes two methods, RNA-seq and NET-seq, to characterize changes in transcription in Spn1-depleted cells, and ChIP-nexus, a high-resolution ChIP-seq method, to characterize chromatin association of specific factors and histone modifications after Spn1 depletion. Aim 2.2 proposes the isolation of mutations that bypass the need for Spn1, to understand the requirements for Spn1 in vivo. Mutations isolated in a pilot study have identified factors required for transcription elongation. These studies will provide new insights into the function of Spn1 within the transcription elongation complex. Together, these studies will greatly advance understanding of Spt5 and Spn1 and thereby increase understanding of transcription and co-transcriptional processes. As Spt5 and Spn1 are conserved, what is learned in studying these model systems will be directly relevant to human biology.